The present invention relates, first, to the HKNG1 gene, shown herein to be associated with central nervous system-related disorders, e.g., neuropsychiatric disorders, in particular, bipolar affective disorder and schizophrenia and with myopia-related disorders. The invention includes recombinant DNA molecules and cloning vectors comprising sequences of the HKNG1 gene, and host cells and non-human host organisms engineered to contain such DNA molecules and cloning vectors. The present invention further relates to HKNG1 gene products, and to antibodies directed against such HKNG1 gene products. The present invention also relates to methods of using the HKNG1 gene and gene product, including drug screening assays, and diagnostic and therapeutic methods for the treatment of HKNG1-mediated disorders, including HKNG1-mediated neuropsychiatric disorders such as bipolar affective disorder, as well as HKNG1-mediated myopia disorders such as early-onset autosomal dominant myopia.
There are only a few psychiatric disorders in which clinical manifestations of the disorder can be correlated with demonstrable defects in the structure and/or function of the nervous system. Well-known examples of such disorders include Huntington""s disease, which can be traced to a mutation in a single gene and in which neurons in the striatum degenerate, and Parkinson""s disease, in which dopaminergic neurons in the nigro-striatal pathway degenerate. The vast majority of psychiatric disorders, however, presumably involve subtle and/or undetectable changes, at the cellular and/or molecular levels, in nervous system structure and function. This lack of detectable neurological defects distinguishes xe2x80x9cneuropsychiatricxe2x80x9d disorders, such as schizophrenia, attention deficit disorders, schizoaffective disorder, bipolar affective disorders, or unipolar affective disorder, from neurological disorders, in which anatomical or biochemical pathologies are manifest. Hence, identification of the causative defects and the neuropathologies of neuropsychiatric disorders are needed in order to enable clinicians to evaluate and prescribe appropriate courses of treatment to cure or ameliorate the symptoms of these disorders.
One of the most prevalent and potentially devastating of neuropsychiatric disorders is bipolar affective disorder (BAD), also known as bipolar mood disorder (BP) or manic-depressive illness, which is characterized by episodes of elevated mood (mania) and depression (Goodwin, et al., 1990, Manic Depressive Illness, Oxford University Press, New York). The most severe and clinically distinctive forms of BAD are BP-I (severe bipolar affective (mood) disorder), which affects 2-3 million people in the United States, and SAD-M (schizoaffective disorder manic type). They are characterized by at least one full episode of mania, with or without episodes of major depression (defined by lowered mood, or depression, with associated disturbances in rhythmic behaviors such as sleeping, eating, and sexual activity). BP-I often co-segregates in families with more etiologically heterogeneous syndromes, such as with a unipolar affective disorder such as unipolar major depressive disorder (MDD), which is a more broadly defined phenotype (Freimer and Reus, 1992, in The Molecular and Genetic Basis of Neurological Disease, Rosenberg, et al., eds., Butterworths, New York, pp. 951-965; McInnes and Freimer, 1995, Curr. Opin. Genet. Develop., 5, 376-381). BP-I and SAD-M are severe mood disorders that are frequently difficult to distinguish from one another on a cross-sectional basis, follow similar clinical courses, and segregate together in family studies (Rosenthal, et al., 1980, Arch. General Psychiat. 37, 804-810; Levinson and Levitt, 1987, Am. J. Psychiat. 144, 415-426; Goodwin, et al., 1990, Manic Depressive Illness, Oxford University Press, New York). Hence, methods for distinguishing neuropsychiatric disorders such as these are needed in order to effectively diagnose and treat afflicted individuals.
Currently, individuals are typically evaluated for BAD using the criteria set forth in the most current version of the American Psychiatric Association""s Diagnostic and Statistical Manual of Mental Disorders (DSM). While many drugs have been used to treat individuals diagnosed with BAD, including lithium salts, carbamazepine and valproic acid, none of the currently available drugs are adequate. For example, drug treatments are effective in only approximately 60-70% of individuals diagnosed with BP-I. Moreover, it is currently impossible to predict which drug treatments will be effective in, for example, particular BP-I affected individuals. Commonly, upon diagnosis, affected individuals are prescribed one drug after another until one is found to be effective. Early prescription of an effective drug treatment, therefore, is critical for several reasons, including the avoidance of extremely dangerous manic episodes, the risk of progressive deterioration if effective treatments are not found, and the risk of substantial side effects of current treatments.
The existence of a genetic component for BAD is strongly supported by segregation analyses and twin studies (Bertelson, et al., 1977, Br. J. Psychiat. 130, 330-351; Freimer and Reus, 1992, in The Molecular and Genetic Basis of Neurological Disease, Rosenberg, et al., eds., Butterworths, New York, pp. 951-965; Pauls, et al., 1992, Arch. Gen. Psychiat. 49, 703-708). Efforts to identify the chromosomal location of genes that might be involved in BP-I, however, have yielded disappointing results in that reports of linkage between BP-I and markers on chromosomes X and 11 could not be independently replicated nor confirmed in the re-analyses of the original pedigrees, indicating that with BAD linkage studies, even extremely high lod scores at a single locus, can be false positives (Baron, et al., 1987, Nature 326, 289-292; Egeland, et al., 1987, Nature 325, 783-787; Kelsoe, et al., 1989, Nature 342, 238-243; Baron, et al., 1993, Nature Genet. 3, 49-55).
Recent investigations have suggested possible localization of BAD genes on chromosomes 18p and 21q, but in both cases the proposed candidate region is not well defined and no unequivocal support exists for either location (Berrettini, et al., 1994, Proc. Natl. Acad. Sci. U.S.A. 91, 5918-5921; Murray, et al., 1994, Science 265, 2049-2054; Pauls, et al., 1995, Am. J. Hum. Genet. 57, 636-643; Maier, et al., 1995, Psych. Res. 59, 7-15; Straub, et al., 1994, Nature Genet. 8, 291-296).
Mapping genes for common diseases believed to be caused by multiple genes, such as BAD, may be complicated by the typically imprecise definition of phenotypes, by etiologic heterogeneity, and by uncertainty about the mode of genetic transmission of the disease trait. With neuropsychiatric disorders there is even greater ambiguity in distinguishing individuals who likely carry an affected genotype from those who are genetically unaffected. For example, one can define an affected phenotype for BAD by including one or more of the broad grouping of diagnostic classifications that constitute the mood disorders: BP-I, SAD-M, MDD, and bipolar affective (mood) disorder with hypomania and major depression (BP-II).
Thus, one of the greatest difficulties facing psychiatric geneticists is uncertainty regarding the validity of phenotype designations, since clinical diagnoses are based solely on clinical observation and subjective reports. Also, with complex traits such as neuropsychiatric disorders, it is difficult to genetically map the trait-causing genes because: (1) neuropsychiatric disorder phenotypes do not exhibit classic Mendelian recessive or dominant inheritance patterns attributable to a single genetic locus, (2) there may be incomplete penetrance, i.e., individuals who inherit a predisposing allele may not manifest disease; (3) a phenocopy phenomenon may occur, i.e., individuals who do not inherit a predisposing allele may nevertheless develop disease due to environmental or random causes; (4) genetic heterogeneity may exist, in which case mutations in any one of several genes may result in identical phenotypes.
Despite these difficulties, however, identification of the chromosomal location, sequence and function of genes and gene products responsible for causing neuropsychiatric disorders such as bipolar affective disorders is of great importance for genetic counseling, diagnosis and treatment of individuals in affected families.
The present invention relates, first, to the discovery, identification, and characterization of novel nucleic acid molecules that are associated with central nervous system-related disorders and processes, e.g., human neuropsychiatric disorders, such as schizophrenia, attention deficit disorder, schizoaffective disorder, dysthymic disorder, major depressive disorder, and bipolar affective disorder (BAD) including severe bipolar affective (mood) disorder (BP-I), bipolar affective (mood) disorder with hypomania and major depression (BP-II). The invention further relates to the discovery, identification, and characterization of proteins encoded by such nucleic acid molecules, or by degenerate, e.g., allelic or homologous, variants thereof. The invention further relates to the discovery, identification, and characterization of novel nucleic acid molecules that are associated with human myopia or nearsightedness, such as early-onset, autosomal dominant myopia, as well as to the discovery, identification, and characterization of proteins encoded by such nucleic acid molecules or by degenerate variants thereof.
In particular, the nucleic acid molecules of the present invention represent, first, nucleic acid sequences corresponding to the gene referred to herein as HKNG1. As demonstrated in the Examples presented below in Sections 6, 8 and 14, the HKNG1 gene is associated with human CNS-related disorders, e.g., neuropsychiatric disorders, in particular BAD. The HKNG1 gene is associated with other human neuropsychiatric disorders as well, such as schizophrenia. As demonstrated in the Example presented below in Section 14, the HKNG1 gene is also associated with human myopia, such as early-onset autosomal dominant myopia.
In addition to the positive correlation between mutations within the HKNG1 gene and individuals exhibiting symptoms of BAD, described in Section 6 and 8, the present invention is further based, in part, on Applicants"" discovery of novel HKNG1 cDNA sequences. Applicants"" discovery of such cDNA sequences has led to the elucidation of the HKNG1 genomic (that is, upstream untranslated, intron/exon, and downstream untranslated) structure, and to the discovery of full-length and alternately spliced HKNG1 variants and the polypeptides encoded by such variants. These discoveries are described in Sections 7 and 10, below. Applicants"" discovery of such cDNA sequences has also led to the elucidation of novel mammalian (e.g., guinea pig and bovine) HKNG1 sequences, and to the discovery of novel allelic variants and polymorphisms of such sequences. These discoveries are described in Section 10 below.
The invention encompasses nucleic acid molecules which comprise the following nucleotide sequences: (a) nucleotide sequences (e.g., SEQ ID NOS: 1, 3, 5, 6, 36, and 37) that comprise a human HKNG1 gene and/or encode a human HKNG1 gene product (e.g., SEQ ID NO: 2; SEQ ID NO: 4), as well as allelic variants, homologs and orthologs thereof, including nucleotide sequences (e.g., SEQ ID NOS:38, 40, 42, 44, and 46-48) that encode non-human HKNG1 gene products (e.g., SEQ ID NOS:39, 41, 43, 45, and 49); (b) nucleotide sequences comprising the novel HKNG1 sequences disclosed herein that encode mutants of the HKNG1 gene product in which sequences encoding all or a part of one or more of the HKNG1 domains is deleted or altered, or fragments thereof; (c) nucleotide sequences that encode fusion proteins comprising a HKNG1 gene product (e.g., SEQ ID NO: 2; SEQ ID NO: 4), or a portion thereof fused to a heterologous polypeptide; and (d) nucleotide sequences within the HKNG1 gene, as well as chromosome 18p nucleotide sequences flanking the HKNG1 gene, which can be utilized, e.g., as primers, in the methods of the invention for identifying and diagnosing individuals at risk for or exhibiting an HKNG1-mediated disorder, such as BAD or schizophrenia, or for diagnosing individuals at risk for or exhibiting a form of myopia such as early-onset autosomal dominant myopia. The nucleic acid molecules of (a) through (d), above, can include, but are not limited to, cDNA, genomic DNA, and RNA sequences.
The invention also encompasses the expression products of the nucleic acid molecules listed above; i.e., peptides, proteins, glycoproteins and/or polypeptides that are encoded by the above HKNG1 nucleic acid molecules.
The compositions of the present invention further encompass agonists and antagonists of the HKNG1 gene product, including small molecules (such as small organic molecules), and macromolecules (including antibodies), as well as nucleotide sequences that can be used to inhibit HKNG1 gene expression (e.g., antisense and ribozyme molecules, and gene or regulatory sequence replacement constructs) or to enhance HKNG1 gene expression (e.g., expression constructs that place the HKNG1 gene under the control of a strong promoter system).
The compositions of the present invention further include cloning vectors and expression vectors containing the nucleic acid molecules of the invention, as well as hosts which have been transformed with such nucleic acid molecules, including cells genetically engineered to contain the nucleic acid molecules of the invention, and/or cells genetically engineered to express the nucleic acid molecules of the invention. In addition to host cells and cell lines, hosts also include transgenic non-human animals (or progeny thereof), particularly non-human mammals, that have been engineered to express an HKNG1 transgene, or xe2x80x9cknock-outsxe2x80x9d that have been engineered to not express HKNG1.
Transgenic non-human animals of the invention include animals engineered to express an HKNG1 transgene at higher or lower levels than normal, wild-type animals. The transgenic animals of the invention also include animals engineered to express a mutant variant or polymorphism of an HKNG1 transgene which is associated with HKNG1-mediated disorder, for example an HKNG1-mediated neuropsychiatric disorders, such as BAD and schizophrenia, or, alternatively, a myopia disorder such as early-onset autosomal dominant myopia. The transgenic animals of the invention further include the progeny of such genetically engineered animals.
The invention further relates to methods for the treatment of HKNG1-mediated disorders in a subject, such as HKNG1-mediated neuropsychiatric disorders and HKNG1-mediated myopia disorders, wherein such methods comprise administering a compound which modulates the expression of a HKNG1 gene and/or the synthesis or activity of a HKNG1 gene product so symptoms of the disorder are ameliorated.
The invention further relates to methods for the treatment of HKNG1-mediated disorders in a subject, such as HKNG1-mediated neuropsychiatric disorders and HKNG1-mediated myopia disorders, resulting from HKNG1 gene mutations or aberrant levels of HKNG1 expression or activity, wherein such methods comprise supplying the subject with a nucleic acid molecule encoding an unimpaired HKNG1 gene product such that an unimpaired HKNG1 gene product is expressed and symptoms of the disorder are ameliorated.
The invention further relates to methods for the treatment of HKNG1-mediated disorders in a subject, such as HKNG1-mediated neuropsychiatric disorders and HKNG1-mediated myopia disorders, resulting from HKNG1 gene mutations or from aberrant levels of expression or activity, wherein such methods comprise supplying the subject with a cell comprising a nucleic acid molecule that encodes an unimpaired HKNG1 gene product such that the cell expresses the unimpaired HKNG1 gene product and symptoms of the disorder are ameliorated.
The invention also encompasses pharmaceutical formulations and methods for treating HKNG1-mediated disorders, including neuropsychiatric disorders, such as BAD and schizophrenia, and myopia disorders, such as early-onset autosomal dominant myopia, involving HKNG1 gene.
In addition, the present invention is directed to methods that utilize the HKNG1 nucleic acid sequences, chromosome 18p nucleotide sequences flanking the HKNG1 human gene and/or HKNG1 gene product sequences for mapping the chromosome 18p region, and for the diagnostic evaluation, genetic testing and prognosis of a HKNG1-mediated disorder, such as a HKNG1-mediated neuropsychiatric disorder or a HKNG1-mediated myopia disorder. For example, in one embodiment, the invention relates to methods for diagnosing HKNG1-mediated disorders, wherein such methods comprise measuring HKNG1 gene expression in a patient sample, or detecting a HKNG1 polymorphism or mutation in the genome of a mammal, including a human, suspected of exhibiting such a disorder. In one embodiment, nucleic acid molecules encoding HKNG1 can be used as diagnostic hybridization probes or as primers for diagnostic PCR analysis for the identification of HKNG1 gene mutations, allelic variations and regulatory defects in the HKNG1 gene which correlate with neuropsychiatric disorders such as BAD or schizophrenia.
The invention still further relates to methods for identifying compounds which modulate the expression of the HKNG1 gene and/or the synthesis or activity of the HKNG1 gene products, including therapeutic compounds, which reduce or eliminate the symptoms of HKNG1-mediated disorders, including HKNG1-mediated neuropsychiatric disorders such as BAD and schizophrenia. In particular, cellular and non-cellular assays are described that can be used to identify compounds that interact with the HKNG1 gene product, e.g., modulate the activity of the HKNG1 and/or bind to the HKNG1 gene product. Such cell-based assays of the invention utilize cells, cell lines, or engineered cells or cell lines that express the HKNG1 gene product.
In one embodiment, such methods comprise contacting a compound to a cell that expresses a HKNG1 gene, measuring the level of HKNG1 gene expression, gene product expression or gene product activity, and comparing this level to the level of HKNG1 gene expression, gene product expression or gene product activity produced by the cell in the absence of the compound, such that if the level obtained in the presence of the compound differs from that obtained in its absence, a compound that modulates the expression of the HKNG1 gene and/or the synthesis or activity of the HKNG1 gene products has been identified.
In another embodiment, such methods comprise contacting a compound to a cell that expresses a HKNG1 gene and also comprises a reporter construct whose transcription is dependent, at least in part, on HKNG1 expression or activity. In such an embodiment, the level of reporter transcription is measured and compared to the level of reporter transcription in the cell in the absence of the compound. If the level of reporter transcription obtained in the presence of the compound differs from that obtained in its absence, a compound that modulates expression of HKNG1 or genes involved in HKNG1-related pathways or signal transduction has been identified.
In yet another embodiment, such methods comprise administering a compound to a host, such as a transgenic animal, that expresses an HKNG1 transgene or a mutant HKNG1 transgene associated with an HKNG1-mediated disorder such as a neuropsychiatric disorder (e.g., BAD or schizophrenia), or to an animal, e.g., a knock-out animal, that does not express HKNG1, and measuring the level of HKNG1 gene expression, gene product expression, gene product activity, or symptoms of an HKNG1-mediated disorder such as an HKNG1-mediated neuropsychiatric disorder (e.g., BAD or schizophrenia). The measured level is compared to the level obtained in a host that is not exposed to the compound, such that if the level obtained when the host is exposed to the compound differs from that obtained in a host not exposed to the compound, a compound modulates the expression of the mammalian HKNG1 gene and/or the synthesis or activity of the mammalian HKNG1 gene products, and/or the symptoms of an HKNG1-mediated disorder such as a neuropsychiatric disorder (e.g., BAD or schizophrenia), has been identified.
The present invention still further relates to pharmacogenomic and pharmacogenetic methods for selecting an effective drug to administer to an individual having a HKNG1-mediated disorder. Such methods are based on the detection of genetic polymorphisms in the HKNG1 gene or variations in HKNG1 gene expression due to, e.g., altered methylation, differential splicing, or post-translational modification of the HKNG1 gene product which can affect the safety and efficacy of a therapeutic agent.
As briefly discussed above, the present invention is based, in part, on the genetic and physical mapping of the HKNG1 gene to a specific portion of the short arm of human chromosome 18 that is associated with human neuropsychiatric disorders, in particular, bipolar affective disorder. These results are described in the Example presented, below, in Section 6. The invention is also based on the elucidation of the HKNG1 nucleotide sequence, amino acid sequence and expression pattern, as described in the Example presented, below, in Section 7. The invention is further based on the identification of specific mutations and/or polymorphisms within the HKNG1 gene which positively correlate with neuropsychiatric disorders, in particular, BAD, as described in the Example presented below in Section 8. These mutations include a point mutation discovered in an individual affected by BAD which is absent from the corresponding wild-type nucleic acid derived from non-affected individuals and linkage disequilibrium of three markers showing cosegregation with a population of individuals with BAD. This mutation is a single base mutation which results in a mutant HKNG1 gene product comprising substitution of a lysine residue for the wild-type glutamic acid residue at HKNG1 amino acid position 202 of the polypeptide of SEQ ID NO:2 or the HKNG1 amino acid residue 184 of the polypeptide of SEQ ID NO:4. These mutations further include the mutations discovered in schizophrenic and BAD patients that are detailed in FIGS. 5A-5C.
As used herein, the following terms shall have the abbreviations indicated.
BAC, bacterial artificial chromosomes
BAD, bipolar affective disorder(s)
BP, bipolar mood disorder
BP-I, severe bipolar affective (mood) disorder
BP-II, bipolar affective (mood) disorder with hypomania and major depression
bp, base pair(s)
EST, expressed sequence tag
HKNG1, Hong Kong new gene 1
lod, logarithm of odds
MDD, unipolar major depressive disorder
ROS, reactive oxygen species
RT-PCR, reverse transcriptase PCR
SSCP, single-stranded conformational polymorphism
SAD-M, schizoaffective disorder manic type
STS, sequence tagged site
YAC, yeast artificial chromosome
xe2x80x9cHKNG1-mediated disordersxe2x80x9d include disorders involving an aberrant level of HKNG1 gene expression, gene product synthesis and/or gene product activity relative to levels found in clinically normal individuals, and/or relative to levels found in a population whose level represents a baseline, average HKNG1 level. While not wishing to be bound by any particular mechanism, it is to be understood that disorder symptoms can, for example, be caused, either directly or indirectly, by such aberrant levels. Alternatively, it is to be understood that such aberrant levels can, either directly or indirectly, ameliorate disorder symptoms, (e.g., as in instances wherein aberrant levels of HKNG1 suppress the disorder symptoms caused by mutations within a second gene).
HKNG1-mediated disorders include, for example, central nervous system (CNS) disorders. CNS disorders include, but are not limited to cognitive and neurodegenerative disorders such as Alzheimer""s disease, senile dementia, Huntington""s disease, amyotrophic lateral sclerosis, and Parkinson""s disease, as well as Gilles de la Tourette""s syndrome, autonomic function disorders such as hypertension and sleep disorders, and neuropsychiatric disorders that include, but are not limited to schizophrenia, schizoaffective disorder, attention deficit disorder, dysthymic disorder, major depressive disorder, mania, obsessive-compulsive disorder, psychoactive substance use disorders, anxiety, panic disorder, as well as bipolar affective disorder, e.g., severe bipolar affective (mood) disorder (BP-I), bipolar affective (mood) disorder with hypomania and major depression (BP-II). Further CNS-related disorders include, for example, those listed in the American Psychiatric Association""s Diagnostic and Statistical manual of Mental Disorders (DSM), the most current version of which is incorporated herein by reference in its entirety.
xe2x80x9cHKNG1-mediated processesxe2x80x9d include processes dependent and/or responsive, either directly or indirectly, to levels of HKNG1 gene expression, gene product synthesis and/or gene product activity. Such processes can include, but are not limited to, developmental, cognitive and autonomic neural and neurological processes, such as, for example, pain, appetite, long term memory and short term memory.